Image Processing Method for a Microscope System

ABSTRACT

An embodiment is disclosed for performing the image processing for analyzing the results of a fluorescence in situ hybridization (FISH) microscopic automated sample analysis to determine specific chromosomal characteristics.

This application is continuation application of U.S. patent applicationSer. No. 11/833,204, filed on Aug. 2, 2007, which claims priority fromU.S. Provisional Application Ser. No. 60/821,536 filed Aug. 4, 2006. Allreferences cited in this specification, and their references, areincorporated by reference herein where appropriate for teachings ofadditional or alternative details, features, and/or technicalbackground.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to image processing methodsemployed for performing automated microscopic analysis for fluorescencein situ hybridization (FISH) detection of genetic characteristics.

2. Description of the Related Art

Conventional optical microscopy generally employs a microscope slide towhich the normal human complement of chromosomes consists of the sexchromosomes (designated X and Y) and 22 autosomes (numbered 1-22). Ithas been estimated that a minimum of 1 in 10 human conceptions has achromosome abnormality. As a general rule, an abnormal number of sexchromosomes is not lethal, although infertility can result. In contrast,an abnormal number of autosomes typically results in early death. Of thethree autosomal trisomies found in live-born babies (trisomy 21, 18 and13), only individuals with trisomy 21 (more commonly known as Downsyndrome), survive past infancy.

Although Down syndrome is easily diagnosed after birth, prenataldiagnosis is problematic. To date, karyotyping of fetal cells remainsthe established method for the diagnosis of Down syndrome and othergenetic abnormalities associated with an aberration in chromosomalnumber and/or arrangement. Such genetic abnormalities include, forexample, chromosomal additions, deletions, amplifications,translocations and rearrangements. The assessment of such abnormalitiesis made with respect to the chromosomes of a healthy individual, i.e.,an individual having the above-described normal complement andarrangement of human chromosomes.

Genetic abnormalities include the above-noted trisomies, such as Downsyndrome, as well as monosomies and disomies. Genetic abnormalities alsoinclude additions and/or deletions of whole chromosomes and/orchromosome segments. Alterations such as these have been reported to bepresent in many malignant tumors. Thus, aberrations in chromosome numberand/or distribution (e.g., rearrangements, translocations) represent amajor cause of mental retardation and malformation syndromes (du Manoiret al., et al., Human Genetics 90(6): 590-610 (1993)) and possibly,oncogenesis. See also, e.g., (Harrison's Principles of InternalMedicine, 12th edition, ed. Wilson et al., McGraw Hill, N.Y., N.Y., pp.24-46 (1991)), for a partial list of human genetic diseases that havebeen mapped to specific chromosomes, and in particular, for a list of Xchromosome linked disorders. In view of the growing number of geneticdisorders associated with chromosomal aberrations, various attempts havebeen reported in connection with developing simple, accurate, automatedassays for genetic abnormality assessment.

In general, karyotyping is used to diagnose genetic abnormalities thatare based upon additions, deletions, amplifications, translocations andrearrangements of an individual's nucleic acid. The “karyotype” refersto the number and structure of the chromosomes of an individual.Typically, the individual's karyotype is obtained by, for example,culturing the individual's peripheral blood lymphocytes until activecell proliferation occurs, preparing single, proliferating (e.g.metaphase, and possibly interphase) cells for chromosome visualization,fixing the cells to a solid support and subjecting the fixed cells to insitu hybridization to specifically visualize discrete portions of theindividual's chromosomes.

The sample contains at least one target nucleic acid, the distributionof which is indicative of the genetic abnormality. By “distribution”, itis meant the presence, absence, relative amount and/or relative locationin one or more nucleic acids (e.g., chromosomes) known to include thetarget nucleic acid. In a particularly preferred embodiment, the targetnucleic acid is indicative of a trisomy 21 and thus, the method isuseful for diagnosing Down syndrome. In a particularly preferredembodiment, the sample intended for Down syndrome analysis is derivedfrom maternal peripheral blood. More particularly, lymphocytes areisolated from peripheral blood according to standard procedures, thecells are attached to a solid support (e.g., by centrifuging onto glassslides), and fixed thereto according to standard procedures (see, e.g.,the Examples) to permit detection of the target nucleic acid.

Nucleic acid hybridization techniques are based upon the ability of asingle stranded oligonucleotide probe to base-pair, i.e., hybridize,with a complementary nucleic acid strand. Fluorescence in situhybridization (“FISH”) techniques, in which the nucleic acid probes arelabeled with a fluorophore (i.e., a fluorescent tag or label thatfluoresces when excited with light of a particular wavelength),represents a powerful tool for the analysis of numerical, as well asstructural aberrations chromosomal aberrations. The method involvescontacting a fixed cell with an antibody labeled with a firstfluorophore for phenotyping the cell via histochemical staining,followed by contacting the fixed cell with a DNA probe labeled with asecond fluorophore for genotyping the cell. The first and secondfluorophores fluoresce at different wavelengths from one another,thereby allowing the phenotypic and genetic analysis on the identicalfixed sample.

Fluorescence in situ hybridization refers to a nucleic acidhybridization technique which employs a fluorophore-labeled probe tospecifically hybridize to and thereby, facilitate visualization of, atarget nucleic acid. Such methods are well known to those of ordinaryskill in the art and are disclosed, for example, in U.S. Pat. No.5,225,326; U.S. patent application Ser. No. 07/668,751; PCT WO 94/02646,the entire contents of which are incorporated herein by reference. Ingeneral, in situ hybridization is useful for determining thedistribution of a nucleic acid in a nucleic acid-containing sample suchas is contained in, for example, tissues at the single cell level. Suchtechniques have been used for karyotyping applications, as well as fordetecting the presence, absence and/or arrangement of specific genescontained in a cell. However, for karyotyping, the cells in the sampletypically are allowed to proliferate until metaphase (or interphase) toobtain a “metaphase-spread” prior to attaching the cells to a solidsupport for performance of the in situ hybridization reaction.

Briefly, fluorescence in situ hybridization involves fixing the sampleto a solid support and preserving the structural integrity of thecomponents contained therein by contacting the sample with a mediumcontaining at least a precipitating agent and/or a cross-linking agent.Exemplary agents useful for “fixing” the sample are well known to thoseof ordinary skill in the art and are described, for example, in theabove-noted patents and/or patent publications.

One fluorescent dye used in fluorescence microscopy is DAPI or4′,6-diamidino-2-phenylindole [CAS number: [28718-90-3], a fluorescentstain that binds strongly to DNA. Since DAPI will pass through an intactcell membrane, it may be used to stain live and fixed cells. DAPI isexcited with ultraviolet light. When bound to double-stranded DNA itsabsorption maximum may be about 358 nm and its emission maximum may beabout 461 nm. DAPI will also bind to RNA, though it is not as stronglyfluorescent. Its emission shifts to about 400 nm when bound to RNA.DAPI's blue emission is convenient for microscopists who wish to usemultiple fluorescent stains in a single sample. There is very littlefluorescence overlap, for example, between DAPI and green-fluorescentmolecules like fluorescein and green fluorescent protein (GFP), orred-fluorescent stains like Texas Red. Other fluorescent dyes are usedto detect other biological structures.

Other types of fluorescing materials are used in fluorescence in situhybridization (FISH). The FISH method uses fluorescent tags to detectchromosomal structure. Such tags may directed to specific chromosomesand specific chromosome regions. Such technique may be used foridentifying chromosomal abnormalities and gene mapping. For example, aFISH probe to chromosome 21 permits one to identify cells with trisomy21, i.e., cells with an extra chromosome 21, the cause of Down syndrome.FISH kits comprising multicolor DNA probes are commercially available.For example, AneuVysion® Multicolor DNA Probe Kit sold by the Vysisdivision of Abbott Laboratories, is designed for in vitro diagnostictesting for abnormalities of chromosomes 13, 18, 21, X and Y in amnioticfluid samples via fluorescence in situ hybridization (FISH) in metaphasecells and interphase nuclei. The AneuVysion® Assay (CEP 18, X, Y-alphasatellite, LSI 13 and 21) Multi-color Probe Panel uses CEP 18/X/Y probeto detect alpha satellite sequences in the centromere regions ofchromosomes 18, X and Y and LSI 13/21 probe to detect the 13q14 regionand the 21q22.13 to 21q22.2 region. The AneuVysion kit is useful foridentifying and enumerating chromosomes 13, 18, 21, X and Y viafluorescence in situ hybridization in metaphase cells and interphasenuclei obtained from amniotic fluid in subjects with presumed high riskpregnancies. The combination of colors emitted by the tags is used todetermine whether there is a normal chromosome numbers or trisomy.

In a similar vein, the UroVysion® kit by the Vysis division of AbbottLaboratories designed to detect chromosomal abnormalities associatedwith the development and progression of bladder cancer by detectinganeuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus viafluorescence in situ hybridization in urine specimens from persons withhematuria suspected of having bladder cancer. The UroVysion Kit consistsof a four-color, four-probe mixture of DNA probe homologous to specificregions on chromosomes 3, 7, 9, and 17. The UroVysion probe mixtureconsists of Chromosome Enumeration Probe (CEP) CEP 3 SpectrumRed, CEP 7SpectrumGreen, CEP 17 SpectrtimAqua and Locus Specific Identifier (LSI9p21) SpectrumGold.

Despite the above-described advances in the development of fluorescentin situ hybridization methods for the diagnosis of geneticabnormalities, the analysis of the fluorophore-labeled sample remainslabor-intensive and involves a significant level of subjectivity. Thisis particularly true in connection with the prenatal diagnosis ofgenetic abnormalities in which fetal cells must either be isolated frommaternal cells or visually distinguished therefrom prior to assessmentfor genetic abnormalities. Thus, for example, a laboratory technicianmust manually prepare and sequentially stain the sample (first, with ahistochemical stain to phenotype the cells, second, with a hybridizationprobe to genotype the cell); visually select fetal cells from othercells in the optical field (using, for example, the above-mentionedhistochemical staining procedure); assess the relative distribution offluorescent color that is attributable to hybridization of thefluorophore-tagged probe; and compare the visually-perceiveddistribution to that observed in control samples containing a normalhuman chromosome complement. As will be readily apparent, theabove-described procedure is quite time-consuming. Moreover, because theresults are visually-perceived, the frequency of erroneous results canvary from one experiment to the next, as well as from one observer tothe next.

The invention disclosed in co-owned U.S. Pat. No. 6,221,607, “Automatedfluorescence in situ hybridization detection of genetic abnormalities,”discloses computer-implemented methods for determining a geneticabnormality such as trisomy 21 which eliminate subjective analysis ofselectively stained chromosomes. More specifically, the patent providesa method for detecting whether a genetic abnormality is present in afixed sample containing at least one target nucleic acid. The method isuseful for diagnosing genetic abnormalities associated with anaberration in chromosomal number and/or arrangement, such as, forexample, chromosomal additions, deletions, amplifications,translocations and rearrangements.

SUMMARY OF THE INVENTION

Embodiments are disclosed which perform various image processingfunctions which may be employed to implement the automated fluorescencein situ hybridization method. The embodiments include an auto-exposuremethod for acceptably imaging all regions of the sample over anintensity range exceeding the dynamic range of the digital electronics;a method for fluorescence in situ hybridization (FISH) object ofinterest enumeration which locates targets within the sample; nucleiidentification which is a method for classifying and characterizing theobjects-of-interest enumerated; segmenting nuclei which, is a method fordefining the shape of an identified object of interest. Embodiments ofthe method are useful to characterize cell nuclei or to enumerate achromosome. An embodiment of the method is adapted for conducting anAneuVysion™ assay (Vysis, Inc., Downers Grove, Ill.).

In an embodiment, there is disclosed:

An image processing method for analyzing a fluorescence in situhybridization image of a fluorescently-hybridized specimen using amicroscope system having a fluorescence exciting light source and anelectronic imaging device, the method comprising the steps of:

illuminating the specimen with fluorescence exciting illumination;

adjusting exposure parameters of the electronic imaging device forcapturing an image of the specimen at a depth of focus within anilluminated field;

enumerating objects of interest in a captured image of the specimen;

identifying a nucleus in the image;

segmenting a nucleus in the image;

counting and characterizing a fluorescent signal occurring within thenucleus; and

interpreting and reporting results.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart representing an overview of an embodiment of acomputer program for carrying out the automated analysis of theinvention.

FIG. 2 is a flow chart representing an embodiment of a program modulefor adjusting exposure parameters.

FIG. 3 is a flow chart representing an embodiment of a program modulefor enumerating objects of interest.

FIG. 4 is a flow chart representing an embodiment of a program modulefor identifying nuclei.

FIG. 5 is a flow chart representing an embodiment of a program modulefor segmenting nuclei.

FIG. 6 is a flow chart representing an embodiment of a program modulefor characterizing signals and reporting results.

DETAILED DESCRIPTION OF THE INVENTION

Auto-Exposure: The acquisition of the digital image typically requiresproper exposure of all of the regions of the specimen being examined.The electronic imaging device may be a multi-pixel planar array oflight-sensitive detectors in a charge coupled device (CCD), orcomplementary metal oxide semiconductor (CMOS) elements, or any othertechnology suitable for converting an optical image into electricalsignals. Exemplary CCD cameras include intensified CCD cameras utilizinggating techniques to achieve gate speeds of less than about ninenanoseconds with improved quantum efficiency (e.g., such as PrincetonInstruments (Trenton, N.J.) PI MAX_(MG) which support a full range of16-bit scientific-grade CCDs), allowing for a very fast response limitedby the time constant of the output phosphor, and electron-bombarded CCD(EBCCD) wherein photons are detected by a photocathode and releasedelectrons are accelerated across a gap and impact on the back side of aCCD (allowing for additional gain and accompanying speed). CMOS camerasin particular may find use in fluorescence microscopy. CMOS cameras havean amplifier and digitizer associated with each photodiode in anintegrated on-chip format. Recent CMOS sensors have a greatly reducedresidual noise, and provide an extraordinary dynamic range.

Low intensity detection can be enhanced through the employment of animage intensifier and similar technologies. The correct exposure timemay be calculated using an algorithm which takes into account conditionssuch as (i) the range of mean image intensity value within certain areas(nuclei), (ii) the range of highest image intensity, (iii) the maximumallowed exposure time, and (iv) independently provided exposureconditions.

Each sensor picture element or pixel typically accumulates an electricalcharge which is in proportion to the number of photons (amount of light)falling on the element. As there is usually an inverse linearrelationship between required exposure time and intensity of the lightimpinging on the pixel, dimmer areas may be imaged in separate exposuresby increasing exposure time. After exposure, the pixels of the imagingarrays can be individually measured in a sequential scanning pattern.Each of the element measurements can then be digitized by means of ananalog to digital converter (A/D) or other means of digitizing the data.The resulting stream of digitized measurements can be stored in a memoryupon which image analysis procedures may be performed.

In most cases, however, a single exposure time does not adequately imageall portions of the specimen since the dynamic range of the image sensorand associated electronics is typically less than the range ofintensities emanating from various locations of a single specimen. Forexample, the dynamic range of an 8 bit D/A converter is 256:1, which maybe inadequate for this application. For that situation, with a singleexposure time, either bright objects or nuclei will be lost if thedimmest objects are properly exposed or, alternatively, dim objects willbe lost when the brightest objects are properly exposed.

When the intensity dynamic range of the imaging field is too large for asingle exposure duration, a multiple exposure strategy can be employed.The portions of the specimen or nuclei with the highest intensities areexposed first using a relatively shorter exposure time, and analyzed.For that exposure, the dimmer areas of the image may be next analyzedusing edge detection or entropy measurement to determine whether alonger exposure time is necessary to adequately image those areas. Ifso, an additional exposure can be made for an appropriately longerexposure time, with the pixel measurements corresponding to the highintensity nuclei excluded or masked from the image. The process may berepeated, for longer exposure times, until no more structures ofinterest are found. As an alternative to a longer exposure time,multiple shorter exposures may be computationally combined to synthesizea single longer exposure. Alternatively, the exposure may be varied bychanging the effective microscope aperture or irradiating intensity.

“Dot” Enumeration: The specimen has finite thickness with objects ofinterest dispersed throughout the depth of the sample. As used herein,an “object of interest” relates to any feature in a microscope fieldthat has been identified as a result of labeling with a FISH probe.Nonlimiting examples of an object of interest include the plasmamembrane or portion thereof, a cytoplasmic organelle or structure, aribosome, a mitochondrion or portion thereof, a mitochondrial nucleicacid, a Golgi membrane, endoplasmic reticulum or portion thereof, anendosome, a nucleus, a nucleolus, a nuclear membrane or portion thereof,a chromosome or portion thereof, and a portion of a DNA molecule.Imaging, therefore, requires that the optical system be individuallyfocused to form well resolved images of each of the objects.Alternatively, a series of exposures may be taken at spaced focal planesselected at sufficiently small intervals so that all objects of interestare acceptably focused. The required number and separation of the focalplanes can readily be determined from the thickness of the specimen andthe depth-of-field of the optical system.

Once the properly focused exposures have been obtained, the images canbe processed to identify and separate the objects of interest orfluorescence in situ hybridization “dots.” These “dots” are revealed bythe fluorescent light that they emit, and the properties of the emittedlight vary in accordance with the characteristics of the object. Inparticular, nonlimiting examples of fluorescent properties emanatingfrom an object of interest include optical properties of the fluorescentlabel, and the intensity of hybridization of the FISH probe to theobject.

Having obtained the images, an enumeration algorithm may be employed toenumerate the fluorescence in situ hybridization objects of interest(“dots”). The first step of the algorithm can be segmentation of the4′,6-diamidino-2-phenylindole (DAPI, a double-stranded DNA stainingfluorescent probe) stained image, in accordance with intensity,effectively defining intensity contours of brightness across each of theimage focal planes. The raw fluorescence in situ hybridization channelimages may be computationally converted into contrast images. Contrastimages are a mathematical transformation of the original image where theintensity of each transformed pixel represents the change in intensityrelative to the adjoining pixels in the original image. Objects withinthe nuclei may be resolved by successively lowering the contrastthreshold from the highest possible value to a preset low value. Foreach object, the highest contrast can be compared to the moving averageand standard deviation of the previous objects. If a significant jump incontrast is detected, all the objects with higher contrast can be markedas potential fluorescence in situ hybridization “dots.” In addition, iftwo potential fluorescence in situ hybridization “dots” are positionedcloser than a preset threshold value, they may be merged to form asingle “dot.” The relative contrasts and sizes of the identifiedpotential fluorescence in situ hybridization “dots” can be compared, andthe final fluorescence in situ hybridization “dots” can be characterizedand logged in a data base.

Nuclei Identification: Once the potential objects of interest orfluorescence in situ hybridization “dots” are identified, automaticpattern recognition techniques may be employed to classify andcharacterize each of the objects. Each of the fluorescence in situhybridization “dot” sites determined by the fluorescence in situhybridization “dot” enumeration analysis can be analyzed to develop anelliptic Fourier shape descriptor of the object that is invariant totranslation, rotation and scaling. Other characterizations, includingbut not limited to object size and emitted intensity distribution withinthe nucleus, may also be employed to describe the object. A patternrecognition algorithm can be employed to identify and categorize theobject of interest based on these characterizations. Initially, thepattern recognition algorithm may be trained by employing an experthuman observer to classify the object and input his result into thepattern recognition data base. After the initial learning period, thealgorithm can be performed automatically and the pattern recognitiondata base continually updated.

Segmenting Nuclei: For each nucleus identified by the patternrecognition algorithm, contours of constant intensity starting atmaximum brightness can be determined. At each point on the respectivecontour, the gradient may also be computed. The size of the nucleus canbe determined, by way of nonlimiting example, as the contourcorresponding to the greatest average gradient.

AneuVysion™ Scanning Method: The AneuVysion™ assay is an FDA clearedtest for prenatal diagnosis which allows for rapid detection of the mostcommon abnormalities of chromosome number using fluorescence in situhybridization. It utilizes molecular genetic techniques to create afluorescent DNA probe that produces a bright microscopic signal when itselectively attaches to one specific part of a particular chromosome.The DNA probes are able to attach to the appropriate chromosomes innon-dividing cells. The signals are different colors for differentchromosomes. By counting the number of signals within a cell, thecytogenetic technologist knows whether either the normal number or atrisomy, monosomy or other aneusomy of the detectable chromosomes ispresent in the fetus.

The disclosed embodiments can be employed to effectively automate theAneuVysion™ assay utilizing the following method. A computationalhistogram may be organized where each bin in the histogram representseach possible combination of chromosomal constituency. The AneuVysion™CEP (Chromosome Enumeration Probe) analysis results utilize a histogramstructure including three coefficients representing the X chromosome,the Y chromosome and chromosome 18. A second histogram structurecorresponding to the LSI probe includes two coefficients representingchromosome 12 and chromosome 13. The automated fluorescence microscopesystem finds a specified number (N) of nuclei using low magnification.In nonlimiting examples, the value of N is specified to be an integersuch as 10, or 20, or 30, or 40, or 50, or 60, or 80, or 100, or 125, or150, or 200, or even more. Furthermore, N may be specified to be aninteger anywhere between the values identified herein. The system thenindividually images each nucleus using high magnification. Thefluorescence in situ hybridization light points are counted for allchannels and the results are organized into the above describedhistogram formulation. The measurement process continues until one bincount reaches a predetermined number (M). For example, the number M maybe 50, corresponding to current government guidelines. More generally,in nonlimiting examples, the value of M is predetermined to be aninteger such as 10, or 20, or 30, or 40, or 50, or 60, or 80, or 100, or125, or 150, or 200, or even more. Furthermore, M may be predeterminedto be an integer anywhere between the values identified herein. If, whenthe N nuclei have been measured, no bin contains the required Mquantity, the system may search for additional nuclei at lowmagnification and continue the measurement at high magnification untilone bin reaches the quantity N. When N is reached, the measurement stopsand the highest bin count may be compared with the next highest bincount. If the highest bin count is some predetermined percentage greaterthan the next greatest, then the result corresponding to that bin can bereported as the clinical result. If it is does not satisfy thecondition, then an inconclusive result can be reported. In variousnonlimiting examples, the predetermined percentage for comparing thehighest bin count with the next highest bin count may be 5%, or 10%, or15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 60%, or70%, or 80%, or 90%, or 100%, or 125%, or 150%, or 175%, or 200%, oreven a higher percent value. Furthermore, the predetermined percentagemay be set at any integral or nonintegral value between those identifiedherein.

An embodiment of the image processing method for analyzing fluorescencein situ hybridization images of the present invention is schematicallyportrayed in FIG. 1. A microscope slide containing a specimen which hasbeen suitably treated to hybridize it in situ to a fluorescent probe isplaced 400 in the field of view of a microscope. In a nonlimitingembodiment the basic elements of a microscope that may be used in thepresent method include an X-Y stage, a mercury or equivalent lightsource suitable to excite fluorescence of the labels, a fluorescencemicroscope, a color detecting CCD image detecting device, a computer,and one or more monitors. The individual elements of the system may becustom-built or purchased off-the-shelf as standard components.Nonlimiting examples of specimens include cells, blood cells, epithelialcells, tissues, disrupted tissues, tissue slices, biopsy samples,excised surgical samples, and the like.

The microscope is adjusted 410 to bring the specimen into focus at afocal plane of choice within the depth of the specimen. The specimen isirradiated 420 with fluorescence exciting illumination causing variousloci of the sample having a labeled probe bound to fluoresce. Theexposure parameters of the electronic imaging device are adjusted toproperly expose these areas 430. As is well known in the art, theexposure parameters may be varied by changing exposure time, and/oraperture, and/or illumination level. An image is captured 440.

In many cases, the intensity range in a field under scrutiny andprovided in a captured image (FIG. 1, 440) will exceed the dynamic rangecapabilities of the electronic imaging device and/or its supportingelectronics. For those situations, referring to FIG. 2, the exposure isadjusted using a sequence of images. The brightest spots are firstidentified 500 and the exposure parameters are set 510 so that thebrightest spots are properly exposed, corresponding to the top end ofthe electronic imaging device's dynamic range. An image is captured 520under these conditions. The less bright portions of the captured imageare analyzed 530 to determine if detail was lost due to underexposure.Underexposure of these areas may not detect dimmer fluorescent spots. Ifthere is a lack of detail in these dimmer regions 540, the exposureparameters are increased 550 to extend the electronic imaging device'sdynamic range into the region below the initial dynamic range. The rangeof the electronic imaging device's supporting electronics is adjusted tocorrespond to the lower intensity levels to be imaged; the output of theimage sensor's pixels corresponding to the initial brightest areas aremasked or bypassed 560 so as not to overload the supporting electronics.A new image is captured 520 and similarly analyzed 530. This process isrepeated until the entire image has been acceptably imaged 570. Theobjects-of-interest are next enumerated from the set of captured images(FIG. 1, 450).

Referring to FIG. 3, a schematic presentation of an enumerationalgorithm, for a given level in the depth of the field of scrutiny thecaptured images are low pass filtered 600. The images may bemathematically re-expressed as contrast images 610 by dividing each ofthe original images by the corresponding low pass filtered version.Objects-of-interest and attributes may be identified by creatingcontours of constant contrast. Contours of successively lower constantcontrast may be resolved 620 by sequentially lowering the contrastthreshold from the highest observed value to a selected low value. Foreach object, the highest contrast may be compared 630 to the movingaverage and the standard deviation of the previous objects. If asignificant jump in contrast is detected 640, all the objects withhigher contrast can be marked as potential objects of interest (“dots”).A “dot” is considered an outlier 650 if it has significantly lowercontrast or lesser size and is excluded from further consideration. Inaddition, if two potential fluorescence in situ hybridization “dots” arepositioned closer than a preset threshold value, they may be merged 660to form a single “dot.” The relative contrasts and sizes of theidentified potential fluorescence in situ hybridization “dots” can becompared, 670 and the final fluorescence in situ hybridization “dots”can be characterized and logged in a data base 680. The process isrepeated (FIG. 1, 455) for additional planes of focus required toperform the analysis throughout the depth of the sample.

Having enumerated the objects-of-interest, the nuclei are thenidentified (FIG. 1, 460). As schematically shown in FIG. 4, a properlyfocused and exposed DAPI image is acquired at low magnification 700.Contours of constant intensity are mathematically generated to segmentthe image, thereby identifying the objects 710. For each of the objectsenumerated 450, a shape characterization such as an elliptic Fouriershape descriptor that is invariant to translation, rotation and scalingis computed 720. The granulometry, or size distribution, of theconstituent features of each object is computed 730. The combination ofall of the variables used to classify the object and characterizationsis employed to fully describe the nuclei. A pattern recognitionalgorithm, in conjunction with an experience based pattern data base, isused to identify and categorize the object-of-interest 740.

The nuclei thus identified are next segmented (FIG. 1, 470) asschematically shown in FIG. 5. A contour of constant intensity,corresponding to the highest intensity, is first computed 800. Thecharacteristics of the objects thereby identified are recorded 810. Thethreshold intensity level is successively reduced and new contours arecomputed 820. As the intensity threshold is lowered, the contourassociated with each of the objects will expand 830, and new objects maybecome visible 840. In some cases, objects which are separated at higherintensity levels may merge as the threshold is reduced 850. Thecomputation of additional contours is terminated once the intensitythreshold level has reached a sufficiently low level 860. A thresholdlevel determination generally uses instrumental or system parametersthat are particular to the microscope system being used. Thusestablishing a threshold is a procedure particular to the installation.By way of nonlimiting example, a microscope system may provide intensitymeasurements as photon counts, or current, or charge accumulation. Aworker of skill in the field of the invention understands and canimplement evaluations of threshold levels that distinguish from overallbackground, on the one hand, and significant contour intensity levels onthe other. At that point, objects which do not conform to the valid sizerange may be eliminated from further consideration 870. The contours ofconstant brightness which have already been computed are used todetermine the edges of each of the objects 880. At each point along eachcontour, the intensity gradient is computed and averaged. The boundaryof each nucleus is defined as the contour having the greatest averagegradient. The objects are thenceforth defined only within their edges890.

Having thus defined and segmented the nuclei (FIG. 1, 470), thefluorescent light signals within each nucleus are counted andcharacterized (FIG. 1, 480), then interpreted and the results reported(FIG. 1, 490). An embodiment of this final step is shown in FIG. 6 andis suitable for use, for example, with an AneuVysion™ assay. Aspreviously described, the AneuVysion™ assay is a test for prenataldiagnosis. The disclosed embodiment described in this application isequally valid for other fluorescence in situ hybridization assays. Amathematical histogram structure is created 900 so that each bincorresponds to a possible combination of fluorescent light spotquantities and colors contained in a nucleus. Each of the first group ofN fluorescent light spots is counted 910, its color is characterized,and the results are added to the appropriate bin of the histogram. Afterthe first N spots have been counted, the maximum count in any of thehistogram bins is determined 920. If that maximum is less than N 930,additional nuclei are located 940. Nonlimiting examples for values of Nhave been disclosed above. If no new nuclei can be located 945, aninconclusive result is reported. The number M is predetermined and maycorrespond to regulatory requirements. A exemplary value for M is 50.Nonlimiting examples for values of M have been disclosed above. Theassociated additional light spots are counted and characterized 950, andthe results are added to the existing bin contents. This processcontinues until the maximum bin count is greater than or equal to N 930.When the maximum number in any bin has reached M, the number in the nextgreatest histogram bin is determined 970. If M exceeds the next greatestnumber by a predetermined Z percent, the chromosomal constituency isreported out 980 as a valid test result. Nonlimiting examples for valuesof a predetermined percent have been disclosed above. If, on the otherhand, M does not exceed the next greatest number by Z percent, the testis reported out as inconclusive 960.

STATEMENT REGARDING PREFERRED EMBODIMENTS

While the invention has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the spirit or scope of the invention as definedby the appended claims. All documents cited herein are incorporated byreference herein where appropriate for teachings of additional oralternative details, features and/or technical background.

1. A computer-usable medium having computer readable instructions storedthereon for execution by a processor to perform a method comprising:adjusting a microscope to bring a specimen into focus at a focal planewithin the depth of the said specimen; irradiating the said specimenwith fluorescence exciting illumination; adjusting the exposureparameters of an electronic imaging device; capturing the image of thesaid specimen with said electronic imaging device; enumeratingobjects-of-interest; identifying nuclei; segmenting said nuclei;counting and characterizing the color of bright fluorescent lightsignals occurring within said nuclei; and interpreting and reporting theresults.